Regenerative materials

ABSTRACT

Methods of making tissue fillers are provided. In certain embodiments, the tissue is flake-like and has regenerative properties.

This application claims priority under 35 U.S.C. §119 to U.S.Provisional Application No. 61/475,378, which was filed on Apr. 14,2011.

The present disclosure relates to tissue fillers, and more particularly,to methods of preparing tissue fillers having a flake-like shape andtissue fillers prepared according to those methods.

Various types of wound dressings and soft tissue fillers are used toregenerate, repair, or otherwise treat diseased or damaged tissues andorgans. Such materials include hyaluronan-based gels, solubilized andcross-linked collagen compositions, micronized tissue matrices, andsynthetic polymeric compositions in hydrogel or other forms. Each ofthese materials has potential drawbacks if used as bulk soft tissuefillers or deep wound dressings, including limited suitability for deepwounds, inability to regenerate, tendency to increase inflammatoryresponse, or tendency to degrade upon exposure to radiation.

According to certain embodiments, a method for preparing a tissue matrixcomposition is provided. The method comprises selecting a collagen-basedtissue matrix, contacting the matrix with a cryoprotectant solution,freezing the matrix, and cutting the matrix, wherein the temperature ofthe tissue matrix ranges from −10° C. to −40° C. for the cutting step.

In certain embodiments, a tissue matrix composition is provided. Thecomposition comprises a collagen-based tissue matrix, wherein the matrixhas been contacted with a cryoprotectant solution and frozen thereafter,and wherein the matrix has been cut at a temperature between −10° C. and−40° C. after freezing.

In certain embodiments, a tissue matrix composition is provided. Thecomposition comprises a collagen-based tissue matrix, wherein the tissuematrix comprises tissue particles having a size distribution rangingfrom 0.2-5 mm in length, 0.203 mm in width, and 0.02-0.3 mm inthickness.

In certain embodiments, a method for preparing a tissue matrixcomposition is provided. The method comprises selecting a collagen-basedmatrix, contacting the matrix with a cryoprotectant solution, freezingthe matrix, adjusting the temperature of the tissue matrix and thecryoprotectant to between −10° C. to −40° C., cutting the frozen tissuematrix, placing the cut tissue matrix in a liquid to form a suspension,and freeze-drying the suspension.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a grating device for performing methods of thepresent disclosure, according to certain embodiments.

FIG. 2 is a diagram of a perfusion column for producing tissue fillersof the present disclosure, according to certain embodiments.

FIG. 3 is a flow chart summarizing the various steps that may be used toproduce tissue fillers, according to certain embodiments of thedisclosed method.

FIGS. 4A and 4B are photographs demonstrating the morphology of varioustissue pieces under light and SEM microscopy respectively, according tocertain embodiments, as described in Example 1.

FIG. 5 is a chart showing the distribution of tissue flake sizeaccording to certain embodiments, as described in Example 1.

FIG. 6 is a chart showing the mass fractions of ice and amorphous tissuedomain as a function of cryoprotectant concentration, according tocertain embodiments, as described in Example 2.

FIG. 7 is a chart showing thermograms of tissue flakes according tocertain embodiments, as described in Example 4.

FIGS. 8A and 8B are charts showing the resistance of processed tissuematerial, according to certain embodiments, to collagenase and trypsindigestion, respectively, as described in Example 4.

FIG. 9A is a photograph of rehydrated tissue flakes, according tocertain embodiments and FIG. 9B is a schematic of a pressure testingpad, as described in Example 5.

FIGS. 10A and 10B are charts showing the width and length sizedistribution, respectively, of processed tissue material, according tocertain embodiments, as described in Example 6.

FIGS. 11A through 11D are photographs of a soft tissue foam, preparedaccording to certain embodiments, as described in Example 6.

FIGS. 12A through 12D show various histological sections of varioustissue matrices after implantation into an animal, according to certainembodiments, as described in Example 6.

DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain exemplary embodimentsaccording to the present disclosure, certain examples of which areillustrated in the accompanying drawings. Wherever possible, the samereference numbers will be used throughout the drawings to refer to thesame or like parts.

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Any range described herein will be understood toinclude the endpoints and all values between the endpoints. It will beunderstood that the benefits and advantages described above may relateto one embodiment or may relate to several embodiments. Whereappropriate, aspects of any of the examples and embodiments describedabove may be combined with aspects of any of the other examplesdescribed to form further examples having comparable or differentproperties and addressing the same or different problems.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

As used herein, “tissue matrix” will refer to material derived fromanimal tissue that includes a collagen-containing matrix. Such tissuematrices can include intact tissues, tissues that have been partially orcompletely decellularized, or synthetic collagenous matrices (e.g., 3-Dmatrices formed from suspended or otherwise processed tissues). Asdescribed further below, suitable tissue matrices can be acellular. Anysuitable tissue matrix can be used, depending on the intendedimplantation site, so long as the tissue is amenable for use with themethods described herein.

The cut tissue matrix, such as the flake-like tissue matrix disclosedherein, possesses several properties that make it suitable to use as abulk soft tissue filler or deep wound dressing. The flake-like tissuematrix is regenerative, permitting host cell repopulation andrevascularization. The large size of the individual tissue flakes,relative to micronized tissue particles, prevents the undesirablemigration of the flakes into the areas surrounding the originaltreatment site. The flake-like tissue matrix disclosed herein has beenfound to be more stable and resistant to enzymatic degradation whencompared to micronized tissue particles. The size and/or geometry of thetissue flakes allows them to form a stable suspension without gelling orphase separation, which makes the tissue fillers suitable for fillinglarge voids (tens to hundreds of milliliters). When used to form asuspension that can be freeze-dried, the suspension of tissue flakes cancontain relatively large, inter-connected channels that permit fluids toflow freely. When used as wound dressings and/or with negative-pressurewound therapy systems, the channels can help support host cellrepopulation and revascularization. These inter-connected channels cantransduce pressure, making the flake-like tissue matrix more amenablefor the treatment of deep wounds, while also being compatible withnegative pressure therapy.

According to certain embodiments, a method for preparing a tissue matrixcomposition is provided. The method comprises selecting a collagen-basedtissue matrix, contacting the matrix with a cryoprotectant solution,freezing the matrix, and cutting the matrix, wherein the temperature ofthe tissue matrix ranges from −10° C. to −40° C. for the cutting step.In certain embodiments, an additional method for preparing a tissuematrix composition is provided. The method comprises selecting acollagen-based matrix, contacting the matrix with a cryoprotectantsolution, freezing the matrix, adjusting the temperature of the tissuematrix and the cryoprotectant to between −10° C. to −40° C., cutting thefrozen tissue matrix, placing the cut tissue matrix in a liquid to forma suspension, and freeze-drying the suspension. In addition, in variousembodiments, tissue matrices produced according to the methods describedherein are provided. In certain embodiments, a tissue matrix compositionis provided. The composition comprises a collagen-based tissue matrix,wherein the matrix has been contacted with a cryoprotectant solution andfrozen thereafter, and wherein the matrix has been cut after freezing ata temperature between −10° C. and −40° C. In certain embodiments, anadditional tissue matrix composition is provided. The compositioncomprises a collagen-based tissue matrix, wherein the tissue matrixcomprises particles having a size distribution ranging from 0.2-5 mm inlength, 0.2-3 mm in width, and 0.02-0.3 mm in thickness.

After a tissue matrix has been selected, a cryoprotectant solution isused to treat the tissue matrix prior to freezing. The cryoprotectantsolution prevent damage to the tissue from damage as a result offreezing and/or thawing, reduce the amount of frozen water in the tissuethrough osmotic dehydration, and can ensure the formation of a highsubzero temperature glassy matrix in the tissue. The use of acryoprotectant can also help maintain a desired balance between icecontent and non-frozen tissue after freezing. A frozen tissue matrixcontaining too much ice can be brittle and difficult to cut. Conversely,frozen tissue matrix with insufficient ice is too soft and warms rapidlyduring cutting, making it difficult to cut as well. Thus, theconcentration of the cryoprotectant in the cryoprotectant solution canbe used to control the ice content and hardness of the frozen matrix. Insome embodiments, the ice content of the frozen matrix (w/w) ranges from40-60% (e.g., 40, 45, 50, 55, or 60%).

Any suitable cryoprotectant can be used in the cryoprotectant solution.In certain embodiments, suitable cryoprotectants can includemaltodextrin, sucrose, polyethylene glycol (PEG), andpolyvinylpyrrolidone (PVP), or combinations thereof. In someembodiments, the cryoprotectant comprises maltodextrin. In someembodiments, the cryoprotectant solution contains 5-50% (w/v)maltodextrin. In some embodiments, the solution contains 15-25% (w/v)maltodextrin.

After treatment with the cryoprotectant solution, the tissue matrix isthen frozen. In certain embodiments, the tissue matrix is frozen at −80°C. Freezing can be accomplished by using a −80° C. freezer. Beforecutting, the temperature of the tissue matrix can be adjusted if neededto a temperature ranging from −10° C. to −40° C. for the cutting step.That temperature range can help maintain the proper balance between icecontent and non-frozen tissue, which facilitates cutting.

After adjusting the temperature, the tissue can be cut. In someembodiments, the tissue is cut into pieces of irregular shape and size.In some embodiments, the irregular shape and size gives the cut tissue aflake-like appearance. In other words, the pieces of cut tissueencompass irregular shapes and sizes, and lack consistency betweenpieces. In some embodiments, the cut tissue has a selected sizedistribution. In certain embodiments, the cut tissue has a sizedistribution ranging from 0.2-5 mm in length, 0.2-3 mm in width, and0.2-0.3 mm in thickness. When hydrated, the majority of cut tissuewithin this size distribution weighs between 0.5 and 2.0 mg. Theirregular shape and size distribution of the cut tissue facilitate theformation of large, interconnected channels that permit body fluid toflow freely when the cut tissue is placed in suspension, and thus aidhost cell repopulation and revascularization. In some uses, the largesize of the cut tissue, relative to smaller micronized tissue particles,prevents migration of the cut tissues into surrounding anatomic siteswhen implanted or placed in or on a wound. Cut tissue that is largerthan the disclosed size distribution may also be undesirable. If the cuttissue is too large, the individual pieces may take up too much spacewhen in suspension and also impede formation of sufficiently largechannels.

Various methods can be used to cut the tissue and still be in accordancewith the disclosed method, provided the tissue is cut into pieces ofirregular shape and size. For example, the tissue matrix can be cutmanually, using scissors. In some embodiments, cutting the tissue matrixcomprises grating the tissue matrix. In some embodiments, a gratingdevice is used to perform the grating step. Any grating device can beused in accordance with the disclosed method, provided it cuts thetissue matrix into pieces of irregular size and shape. In someembodiments, the grating device is a grater, such as a MICROPLANE®grater. In other embodiments, the grating device is a grating wheel.

In certain embodiments, the cutting step can be automated. Automatingreduces the amount of time necessary to prepare the cut tissue andfacilitates cutting larger amounts of tissue. In various embodiments,one or more aspects of the cutting process can be automated. Forexample, the grating device itself can be automated so that manualeffort is no longer required to cut the tissue matrix. It is alsopossible to automate delivery of the tissue matrix to the cuttingdevice. Further, it is possible to automate removal of tissue matrixonce it has been cut. One example of an automated cutting apparatus isillustrated in FIG. 1. As shown, the apparatus comprises a grating wheel101 that is fitted with blades 102 for cutting a matrix 107. Below thegrating wheel is a collection tray 103. The grating wheel 101 andcollection tray are enclosed in a housing unit 104, which protects theoperator from direct contact with the blades 102. The housing unit 104is fitted with a loading bay 106. In this example, the loading bay 106is inclined so that any samples loaded onto it will move toward thegrating wheel 101 by gravity. Tissue matrix 107 is placed onto theloading bay 106 and moves toward the grating wheel 101. The blades 102of the grating wheel 101 cut the tissue matrix 107 into flakes 108 whichfall into the collection tray 103 below.

In various embodiments, the disclosed methods can further compriseadditional processing before or after cutting. For example, in variousembodiments, the disclosed methods comprise use of a pre-processedacellular tissue matrix, which is described in more detail below. Inother embodiments, an intact, cellular tissue may be used, which can befurther processed to produce a suitable acellular tissue matrix. Thefurther processing may comprise decellularization, DNA removal, andremoval of α-gal epitopes or other antigens. Decellularization, DNAremoval, and α-gal antigen removal are described in further detailbelow. Further processing of the cut tissue may include disinfecting thetissue matrix. In some embodiments, the flake-like tissue matrix isdisinfected with isopropyl alcohol (IPA) (e.g., at about 70% IPA).

Processing of the tissue flakes is also amenable to automation.Automation can include any process that does not require the manualdelivery and removal of the solutions that facilitate processing.Automation of tissue processing reduces time spent manually replacingprocessing solutions and minimizes operator handling of the tissueflakes. In some embodiments, automation of tissue processing comprisesuse of a closed, low-pressure perfusion column. Decellularization, DNAremoval, α-gal antigen removal, and disinfection can all be performedwithin the column, which allows for high-throughput processing of thecut tissue. Cut tissue matrix is loaded into the column, and air ispurged from the column. Decellularization, DNA removal, α-gal antigenremoval, and disinfection are achieved by stepwise perfusion of theappropriate solutions at or near atmospheric pressure. Detergent, DNAse,and α-galactosidase solutions are described in more detail below.

An example of an automated processing system is shown in FIG. 2. Theprocessing system comprises a solution pumping system 201 into whichvarious processing solutions are placed. The pumping system 201 sends anappropriate solution through tubing 202 into a closed perfusion column204, which has been fitted with a process solution inlet 203 and outlet205. The appropriate solution then contacts the tissue flakes 108 insidethe perfusion column 204 and then leaves the column 204 through tubing202 connected to the process solution outlet 205 on one end and thesolution pumping system 201 on the other end.

The disclosed tissue fillers can be further treated to producesubstantially aseptic or sterile materials. Accordingly, in variousembodiments, the tissue fillers can be sterilized after preparation. Asused herein, a “sterilization process” can include any process thatreduces bioburden in a sample, but need not render the sample absolutelysterile.

Certain exemplary processes include, but are not limited to, a gammairradiation process, an e-beam irradiation process, ethylene oxidetreatment, and propylene oxide treatment. Suitable sterilizationprocesses include, but are not limited to, those described in, forexample, U.S. Patent Publication No. 2006/0073592A1, to Sun et al.; U.S.Pat. No. 5,460,962 to Kemp; U.S. Patent Publication No. 2008/0171092A1,to Cook et al. In some embodiments, sterilization is performed inconjunction with packaging of the flakes, while in other embodiments,sterilization can occur after packaging.

After the tissue flakes are prepared by the disclosed methods, they maybe stored for some time before implantation in or on a patient. Incertain embodiments, the tissue filler may be packaged in a Tyvek pouchfor storage purposes. The tissue filler may also be stored in differentstates. In some embodiments, the flake-like tissue filler isfreeze-dried after preparation. In certain embodiments, freeze-drying ofthe flake-like tissue filler is performed before or during packaging. Incertain embodiments, the tissue flakes are stored in a hydrated state.The tissue flakes can be hydrated in various solutions, for example, anaqueous preservation solution.

The various steps described above can be combined, added, deleted, orotherwise modified as necessary. For example, if the starting materialis a porcine hide, removing the epidermis and subcutaneous fat may benecessary before cutting. However, removal of those components is notrequired if the starting material is an acellular tissue matrix.Further, if one is using porcine hide as the starting material ratherthan an acellular tissue matrix, additional processing may be necessaryafter cutting.

A sample protocol in accordance with the disclosed methods is providedin FIG. 3, using porcine hides as the starting material. Specificdetails regarding each step are provided throughout the presentdisclosure. Fresh porcine hides are collected with the hairssubsequently removed 301. The epidermis and subcutaneous fat layers arethen removed, leaving the dermal tissue 302. The dermal tissue is thenincubated in a cryoprotectant solution prior to freezing to maintain theproper balance between ice content and amorphous tissue during cutting303. After incubation in the cryoprotectant solution, the tissue isfrozen, brought to a temperature between −10° C. and −40° C., and cut toa desired size and shape. The flakes are then incubated in anappropriate solution to decellularize the tissue 305. The tissue is thentreated with treated with enzymes to remove DNA and α-gal epitopes 306.After enzyme treatment, the flakes are disinfected with using an IPAsolution 307 and washed in buffer 308.

After washing, the tissue flakes are packaged for storage. Packaging canbe performed in conjunction with freeze-drying 309 or with sterilization312. If the flakes are freeze-dried in conjunction with packaging 309,they are then sterilized 310, resulting in sterile, freeze-dried flakes311. Alternatively, if packaging and sterilization are performedtogether 312, the flakes can be freeze-dried after 314, which alsoresults in sterile, freeze dried flakes 311. The flakes can also behydrated after the packaging and sterilization step 312, resulting insterile, hydrated tissue flakes 313.

The disclosed tissue matrix composition can be used in various ways. Incertain embodiments, the tissue matrix composition can form a stable,non-gelling, low density suspension. In certain embodiments, thedisclosed tissue matrix composition can be used as a bulk tissue fillerfor tissue regeneration and repair. Methods of treatment using thecomposition include selecting an anatomical site for treatment andimplanting the tissue filler into the treatment site. Examples includethe direct application of the flake-like tissue material to deep woundsand large soft tissue voids that may occur during certain types ofsurgeries, such as lumpectomies. The tissue filler may be also be usedin the treatment of pressure ulcers, diabetic foot ulcers, or periostealbone defects. The tissue filler can also be used for reconstructingfacial features as well as correcting facial defects, includingtreatment of wrinkles, skin loss, or skin atrophy. In other embodiments,the flake-like tissue material may be used as a carrier for controlleddelivery of other bioactive substances. Examples of bioactive substancesinclude, but are not limited to, antimicrobial agents, cytokines, growthfactors, and drugs. Bioactive substances can also includenon-collagenous tissue, such as adipose tissue, or cells, including stemcells. In certain embodiments, tissue flakes that have been subsequentlyfreeze-dried can be hydrated in solutions that contain bioactivesubstances, and then applied to the sites as needed. In otherembodiments, the flake-like tissue filler can be applied as a slurry. Ifan aqueous suspension of tissue flakes is blended briefly (30 to 300seconds, for example), it becomes a loosely intertwined fibrillar slurrythat is flowable for convenient application.

In certain instances, a tissue foam may be desired. Depending on thesurgical procedures and particular circumstances of tissue repair, useof a tissue foam may be appropriate. Tissue foams can be used to treatwounds or damaged tissues that are not defined by voids with aparticular boundary. For example, one could use surgical adhesives orsutures to attach a tissue foam to tissues or organs. In other cases,tissue flakes may be more desirable when there is a need to fill voidsof any shape, which are defined by a particular boundary. For example,tissue flakes are suitable when tumors are removed via laproscopicprocedures or cryosurgery, due to the small openings that result. Tissuefoams can also be made to have specific sizes and shapes, such assheets, spheres, and cubes, for certain well-defined surgeries. Forexample, tissue foam sheets can be used to partially or completely coversurgical implants to reduce the dramatic effect of initial implant/bodyinteractions and potentially slow capsule formation. Further, tissuefoams can be used as components of negative pressure wound therapysystems, such as the VAC® system, which is produced by Kinetic Concepts,Inc. Such systems can be used to treat a variety of tissues sites andinclude, for example, a negative pressure source such as a pump and oneor more treatment materials, which often include a porous foam ormanifold. General examples of such systems are described in U.S. PatentPublication Number, 2010/0040687 A1, which was filed on Aug. 13, 2009.

The use of the disclosed tissue flakes facilitates the preparation oftissue foam in several ways. The tissue flakes have a small mass butlarge surface area, which facilitates further processing, and the flakesare amenable to preparing a uniform fiber suspension. In contrast tousing ground or micronized tissue particles or fibers, tissue flakes areunlikely to gel or cake during decellularization. Finally, the processof preparing the tissue flakes first adds an additional layer of sizereduction, which assists in the preparation of a uniform suspension.

Accordingly, the methods described herein can also be used to prepare aregenerative foam using the cut tissue described above as the startingmaterial. As discussed above, a tissue matrix is selected and acryoprotectant is used to treat the tissue matrix prior to freezing. Thetissue matrix is then frozen and cut at a temperature ranging from −10°C. to −40° C. after freezing. As before, a pre-processed, acellulartissue matrix may be used in conjunction with the disclosed method. Inother embodiments, the tissue matrix can be decellularized after cuttingas described above.

The cut tissue matrix is then placed in a liquid to form a suspension.Any suitable liquid may be used, provided it does not interfere with theregenerative properties of the tissue matrix. In some embodiments, thecut tissue matrix is placed in an aqueous solution. After being placedin solution, the tissue matrix can be mixed within the solution. Invarious embodiments, the solution is mixed until a stable tissuesuspension is formed, and/or until the tissue size distribution reachesa desired level. Mixing can be accomplished by any suitable means thatachieve these ends, such as agitating, shaking, or vortexing the tissuematrix once it is in solution. Blending can also be used to mix thetissue matrix in solution. In certain embodiments, a blender is used tomix the matrix. Mixing may also be achieved by using a pressure jet, anultrasound device, or a combination of the two. For example, afterdecellularization, tissue flakes in solution can go through apressurized nozzle, where the tissue flakes break into fibers. One canalso place tissue flakes in solution into an ultrasonic field to breakthe tissue flakes into a fiber suspension. A sonic nozzle can also beused, which combines ultrasound and pressure to break the tissue flakesinto a fibrous suspension. The consistency of the suspension can becontrolled by how much cut tissue matrix is added to the liquid. In someembodiments, the amount of cut tissue matrix in the liquid ranges from20-40% (w/v). In certain embodiments, the amount of cut tissue matrix inthe liquid is 25% (w/v).

After formation of a suspension, the tissue suspension is thenfreeze-dried. Freeze-drying can be performed under aseptic conditions toprevent contamination of the tissue matrix. The tissue suspension canalso be aliquoted into an appropriate container, so that uponfreeze-drying, the tissue matrix will be cast in the desired shape.

The process of mixing and freeze-drying results in a tissue compositionwherein small tissue filaments of various dimensions are intertwined andinterlocked with one another. In certain embodiments, the process ofmixing and freeze-drying the tissue suspension results in a tissue foam.A foam can be produced, for example, by blending an aqueous suspensionfreeze-dried tissue flakes in a blender. After processing, the tissuecomposition contains interconnected macropores due to the intertwinedpieces of small tissue, which permit the free flow of fluid and helpsupport cell repopulation and revascularization.

The tissue foam disclosed herein can be further processed as needed. Thetissue foam can be disinfected or sterilized as described above. Thefreeze-dried material can also be further treated to increase thestrength of the tissue foam using heat and vacuum conditions. Withoutbeing bound to theory, strengthening of the tissue foam may occurthrough physical interlocking, biochemical cross-linking, or acombination of the two. Physically, final dehydration results in surfacetension, which pulls tissue fragments closer together and forms hydrogenbonds between the hydroxyl groups of adjacent collagen fibers.Chemically, the treatment results in amide formation between carboxyland amino groups, as well as esterification and glycation of collagenand other extracellular matrix protein amino groups. In someembodiments, the heat applied to the tissue matrix is limited to avoiddenaturation of the dry proteins contained in the tissue matrix. Dryproteins in the tissue matrix typically denature between 130° C. and170° C. In one embodiment, the strength of the tissue foam can beincreased by treating the freeze-dried material at a temperature above30° C. but below the denaturation temperatures listed above under vacuumconditions. In some embodiments, the strength of the tissue foam can beincreased by treating the freeze-dried material at approximately 100° C.under vacuum for a certain period of time. In some embodiments, thedried material may be treated under for vacuum for 24 hours. Othersuitable periods of time can be readily identified and tested by thoseskilled in the art.

After processing, the tissue foam may be stored for some time beforeimplantation in or on a patient. In certain embodiments, the tissue foammay be packaged in a Tyvek pouch for storage purposes. The tissue foamitself may also be stored in different states. In some embodiments, thetissue foam is stored in its already freeze-dried state. In otherembodiments, the tissue foam is stored in a hydrated state.

Tissue Matrices

As noted above, the methods described herein can be used to produceflake-like tissue fillers using a variety of different tissue types, solong as the tissue includes a collagen-containing matrix amenable foruse with the methods described above. Such tissue matrices can includeintact tissues, tissues that have been partially or completelydecellularized, or synthetic collagenous matrices (e.g., 3-D matricesformed from suspended or otherwise processed tissues).

The tissue matrix can be produced from a range of tissue types. Forexample, the tissue matrix can be derived from fascia, pericardialtissue, dura, umbilical tissue, placental tissue, cardiac valve tissue,ligament tissue, tendon tissue, arterial tissue, venous tissue, neuralconnective tissue, urinary bladder tissue, ureter tissue, and intestinaltissue. In other embodiments, the tissue matrix comprises a dermaltissue matrix. In certain embodiments, the tissue matrix comprisesporcine dermal matrix.

In certain embodiments, the tissues can include a mammalian soft tissue.For example, in certain embodiments, the tissue can include mammaliandermis. In certain embodiments, the dermis can be separated fromsurrounding epidermis and/or other tissues, such as subcutaneous fat. Incertain embodiments, the tissue sample can include small intestinesubmucosa. In certain embodiments, the tissue samples can include humanor non-human sources. Exemplary, suitable non-human tissue sourcesinclude, but are not limited to, pigs, sheep, goats, rabbits, monkeys,and/or other non-human mammals.

The tissue matrices can be implanted at a variety of different anatomicsites. For example, tissue matrices can be implanted around breastimplants; around or replacing vascular structures; around or replacingluminal structures (e.g., ureters, nerves, lymphatic tissues,gastrointestinal structures); on or replacing heart valves, pericardium,or other cardiac structures; in or on bony or cartilaginous materials(e.g., ears, noses, articular surfaces, around dental structures, oralong any short of long bone); and/or surrounding, lining, supporting,or replacing any body cavity (e.g., bladder, stomach).

Tissue matrices can be selected to provide a variety of differentbiological and mechanical properties. For example, an acellular tissuematrix can be selected to allow tissue ingrowth and remodeling to assistin regeneration of tissue normally found at the site where the matrix isimplanted. For example, an acellular tissue matrix, when implanted on orinto fascia, may be selected to allow regeneration of the fascia withoutexcessive fibrosis or scar formation. In certain embodiments, the tissuematrix can be formed from ALLODERM® or STRATTICE™, which are human andporcine acellular dermal matrices, respectively. Alternatively, othersuitable acellular tissue matrices can be used, as described furtherbelow.

In some embodiments, the collagen-based material comprises an acellulartissue matrix. In certain embodiments, these matrices can be completelydecellularized to yield acellular tissue matrices to be used forpatients. For example, various tissues, such as skin, intestine, bone,cartilage, nerve tissue (e.g., nerve fibers or dura), tendons,ligaments, or other tissues can be completely decellularized to producetissue matrices useful for patients. Suitable processes for producingacellular tissue matrices are described below.

In general, the steps involved in the production of an acellular tissuematrix include harvesting the tissue from a donor (e.g., a human cadaveror animal source) and cell removal under conditions that preservebiological and structural function. In certain embodiments, the processincludes chemical treatment to stabilize the tissue and avoidbiochemical and structural degradation together with or before cellremoval. In various embodiments, the stabilizing solution arrests andprevents osmotic, hypoxic, autolytic, and proteolytic degradation,protects against microbial contamination, and reduces mechanical damagethat can occur with tissues that contain, for example, smooth musclecomponents (e.g., blood vessels). The stabilizing solution may containan appropriate buffer, one or more antioxidants, one or more oncoticagents, one or more antibiotics, one or more protease inhibitors, and/orone or more smooth muscle relaxants.

The tissue is then placed in a decellularization solution to removeviable cells (e.g., epithelial cells, endothelial cells, smooth musclecells, and fibroblasts) from the structural matrix without damaging thebiological and structural integrity of the collagen matrix. Thedecellularization solution may contain an appropriate buffer, salt, anantibiotic, one or more detergents (e.g., TRITON X-100™, sodiumdeoxycholate, polyoxyethylene (20) sorbitan mono-oleate), one or moreagents to prevent cross-linking, one or more protease inhibitors, and/orone or more enzymes. In some embodiments, the decellularization solutioncomprises 1% TRITON X-100™ in RPMI media with Gentamicin and 25 mM EDTA(ethylenediaminetetraacetic acid). In some embodiments, the tissue isincubated in the decellularization solution overnight at 37° C. withgentle shaking at 90 rpm. For example, in some embodiments, 2% sodiumdeoxycholate is added to the decellularization solution.

After the decellularization process, the tissue sample is washedthoroughly with saline. In some exemplary embodiments, e.g., whenxenogenic material is used, the decellularized tissue is then treatedovernight at room temperature with a deoxyribonuclease (DNase) solution.In some embodiments, the tissue sample is treated with a DNase solutionprepared in DNase buffer (20 mM HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 20 mM CaCl₂ and 20mM MgCl₂). Optionally, an antibiotic solution (e.g., Gentamicin) may beadded to the DNase solution. Any suitable buffer can be used as long asthe buffer provides suitable DNase activity.

While an acellular tissue matrix may be made from the same species asthe acellular tissue matrix graft recipient, different species can alsoserve as tissue sources. Thus, for example, an acellular tissue matrixmay be made from porcine tissue and implanted in a human patient.Species that can serve as recipients of acellular tissue matrix anddonors of tissues or organs for the production of the acellular tissuematrix include, without limitation, mammals, such as humans, nonhumanprimates (e.g., monkeys, baboons, or chimpanzees), pigs, cows, horses,goats, sheep, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats,or mice.

Elimination of the α-gal epitopes from the collagen-containing materialmay diminish the immune response against the collagen-containingmaterial. The α-gal epitope is expressed in non-primate mammals and inNew World monkeys (monkeys of South America) as well as onmacromolecules such as proteoglycans of the extracellular components. U.Galili et al., J. Biol. Chem. 263:17755 (1988). This epitope is absentin Old World primates (monkeys of Asia and Africa and apes) and humans,however. Id. Anti-gal antibodies are produced in humans and primates asa result of an immune response to α-gal epitope carbohydrate structureson gastrointestinal bacteria. U. Galili et al., Infect. Immun. 56:1730(1988); R. M. Hamadeh et al., J. Clin. Invest. 89:1223 (1992).

Since non-primate mammals (e.g., pigs) produce α-gal epitopes,xenotransplantation of collagen-containing material from these mammalsinto primates often results in immunological activation because ofprimate anti-Gal antibodies binding to these epitopes on thecollagen-containing material. U. Galili et al., Immunology Today 14:480(1993); M. Sandrin et al., Proc. Natl. Acad. Sci. USA 90:11391 (1993);H. Good et al., Transplant. Proc. 24:559 (1992); B. H. Collins et al.,J. Immunol. 154:5500 (1995). Furthermore, xenotransplantation results inmajor activation of the immune system to produce increased amounts ofhigh affinity anti-gal antibodies. Accordingly, in some embodiments,when animals that produce α-gal epitopes are used as the tissue source,the substantial elimination of α-gal epitopes from cells and fromextracellular components of the collagen-containing material, and theprevention of re-expression of cellular α-gal epitopes can diminish theimmune response against the collagen-containing material associated withanti-gal antibody binding to α-gal epitopes.

To remove α-gal epitopes, after washing the tissue thoroughly withsaline to remove the DNase solution, the tissue sample may be subjectedto one or more enzymatic treatments to remove certain immunogenicantigens, if present in the sample. In some embodiments, the tissuesample may be treated with an α-galactosidase enzyme to eliminate α-galepitopes if present in the tissue. In some embodiments, the tissuesample is treated with α-galactosidase at a concentration of 300 U/Lprepared in 100 mM phosphate buffer at pH 6.0. In other embodiments, theconcentration of α-galactosidase is increased to 400 U/L for adequateremoval of the α-gal epitopes from the harvested tissue. Any suitableenzyme concentration and buffer can be used as long as sufficientremoval of antigens is achieved.

Alternatively, rather than treating the tissue with enzymes, animalsthat have been genetically modified to lack one or more antigenicepitopes may be selected as the tissue source. For example, animals(e.g., pigs) that have been genetically engineered to lack the terminalα-galactose moiety can be selected as the tissue source. Fordescriptions of appropriate animals see co-pending U.S. application Ser.No. 10/896,594 and U.S. Pat. No. 6,166,288, the disclosures of which areincorporated herein by reference in their entirety. In addition, certainexemplary methods of processing tissues to produce acellular matriceswith or without reduced amounts of or lacking alpha-1,3-galactosemoieties, are described in Xu, Hui. et al., “A Porcine-Derived AcellularDermal Scaffold that Supports Soft Tissue Regeneration: Removal ofTerminal Galactose-α-(1,3)-Galactose and Retention of Matrix Structure,”Tissue Engineering, Vol. 15, 1-13 (2009), which is incorporated byreference in its entirety.

After the acellular tissue matrix is formed, histocompatible, viablecells may optionally be seeded in the acellular tissue matrix to producea graft that may be further remodeled by the host. In some embodiments,histocompatible viable cells may be added to the matrices by standard invitro cell co-culturing techniques prior to transplantation, or by invivo repopulation following transplantation. In vivo repopulation can beby the recipient's own cells migrating into the acellular tissue matrixor by infusing or injecting cells obtained from the recipient orhistocompatible cells from another donor into the acellular tissuematrix in situ. Various cell types can be used, including embryonic stemcells, adult stem cells (e.g. mesenchymal stem cells), and/or neuronalcells. In various embodiments, the cells can be directly applied to theinner portion of the acellular tissue matrix just before or afterimplantation. In certain embodiments, the cells can be placed within theacellular tissue matrix to be implanted, and cultured prior toimplantation.

Although general process parameters for production of acellular tissuematrices are described, a variety of collagen-containing acellularmaterials are available, and the methods of processing such materials toproduce flake-like tissue fillers may be used with any of thosematerials. For example, a number of biological scaffold materials aredescribed by Badylak et al., and the methods of the present disclosurecan be used to produce flake-like tissue fillers using any of thosematerials, or any other similar materials. Badylak et al.,“Extracellular Matrix as a Biological Scaffold Material Structure andFunction,” Acta Biomaterialia (2008), doi:10.1016/j.actbio.2008.09.013.

Example 1 Cryopreservation and Cryocutting of Split Porcine Dermis

Fresh porcine hides were collected, and the hair was removed. Dermistissue was obtained from the hides by splitting off the epidermis layerand subcutaneous fat (hypodermis) layer. The porcine dermis was rinsedwith Dulbecco's phosphate-buffered saline (PBS), and incubated for 4 to6 hours in a cryoprotectant solution containing 50 mM sodium phosphate,10 mM ethylenediamine tetraacetic acid (EDTA) and 35% (w/v) maltodextrin(pH 7.0). Cryoprotectant-treated dermis sheets were then frozen in a−80° C. freezer. Frozen dermis tissue was grated into tissue flakes byusing a medium-sized MICROPLANE® grater (grater openings areapproximately 2.2 to 3.2 mm wide and 0.2 mm thick).

The tissue flakes were then washed for 10-15 minutes in PBS to removethe cryoprotectant and then fixed in 2% glutaraldehyde solution. Themorphology of the tissue flakes was then observed using light microscopyand scanning electron microscopy (SEM). For examination under lightmicrocopy, tissue samples were stained with Sirius Red. For SEMexamination, tissue samples were dehydrated stepwise in ethanolsolutions of increasing concentration (25%, 50%, 75%, 90%, 95%, 98% and100%), dried with supercritical CO₂ in a critical point dryer, andsputter-coated with gold. Tissue specimens were viewed under high vacuumand low electron voltage (5 kV). As shown in FIGS. 4A and 4B, themorphology of the cut pieces was observed to be flake-like under bothtypes of microscopy.

To determine the size (mass) distribution of the tissue flakes,glutaraldehyde-fixed tissue pieces were washed in distilled water toremove glutaraldehyde residue. The wet weight of greater than 200 tissuepieces was determined after blot-drying surface water with surgicalgauze. FIG. 5 shows the mass-based size distribution of the tissueflakes. More than 70% of the tissue fragments weighed between 0.5 and2.0 mg, with an average weight of 1.3 mg. Few tissue pieces weighed over4 mg. The tissue fragment size was in the expected range after using amedium microplane (2 to 3 mm wide and 0.2 mm thick, nominal tissuevolume of 0.8 to 1.8 mm³).

Example 2 Control of Ice Content in Frozen Porcine Dermis

The proper ratio between ice and amorphous phase frozen dermis tissuefacilitates cutting dermis sheets into a flake-like tissue matrix.Frozen dermis sheets that were not treated in cryoprotectant solutioncontained too much ice, were too brittle, and were difficult to cut. Onthe other hand, frozen dermis sheets with low ice content were soft andwarmed rapidly during cutting, also making them difficult to cut. Theice content and the hardness of the frozen dermis sheets were controlledby pretreating the dermis sheets in cryoprotectant solutions thatreduced ice formation while facilitating formation of an amorphoustissue matrix with a high subzero glass transition temperature.

Split porcine dermis was prepared as described in Example 1.Maltodextrin solutions containing 0% to 35% (w/v) maltodextrin were madewith phosphate buffer solution (50 mM sodium phosphate, 10 mM EDTA, pH7.0). Samples of split porcine dermis were incubated in thecryoprotectant solutions overnight. After incubation, water andcyroprotectant content in dermis tissue samples were determined. Icecontents of frozen tissue samples were determined by differentialscanning calorimetry according to their ice melting enthalpies. FIG. 6shows the mass fractions of ice and amorphous tissue domain in thefrozen tissue pretreated in different cryoprotectant solutions. In theabsence of maltodextrin (PBS buffer alone), porcine dermis had 75% (w/v)water and 25% dry mass. Upon freezing, ˜93% of dermis water crystallizedas ice, resulting in a mass fraction of 70% ice and a mass fraction of30% freeze-concentrated amorphous (non-frozen) domain. The correspondingvolume fractions were 77% and 23% for ice and non-frozen domain,respectively. With increasing maltodextrin concentrations, the icefraction of incubated porcine dermis tissue decreased and the fractionof the non-frozen domain increased. For porcine dermis incubated in 35%maltodextrin solution, the mass fractions of ice and non-frozen domainwere reduced to 34% and increased to 66%, respectively (correspondingvolume fractions of 42% and 58%, respectively). Reduced ice formationwas due to both osmotic tissue dehydration and maltodextrin penetrationinto the extracellular matrix of the dermis tissue. As a result ofmaltodextrin penetration into the dermis extracellular matrix, the glasstransition temperature of the frozen dermis amorphous domain increasedto −17° C. Porcine tissue treated with 15-35% maltodextrin solutionscould be easily grated into tissue flakes.

Example 3 Processing of Dermal Tissue Flakes

Tissue flakes prepared as described in Example 1 were further processedto remove cellular components and α-galactosyl epitopes. The processincluded the following steps: (i) rinsing off the cryoprotectants, (ii)decellularization, (iii) enzyme treatment, (iv) disinfection, (v) freezedrying, (vi) sterilization, and (vii) secondary packaging.

(i) Removal of cryoprotectants. Tissue flakes were placed into 225 mLconical centrifuge bottles (˜30 g/bottle). The material was washed with150 mL of sterile PBS for about 10 minutes. The tissue suspension wascentrifuged at 500 rpm for 3 minutes, and the supernatant was discarded.The tissue pellet was re-suspended with 150 mL sterile distilled water,and centrifuged again at 500 rpm for 3 minutes to collect the tissueflakes.

(ii) Decellularization. Tissue material was decellularized at ambienttemperature with agitation in 150 ml of 2% (w/v) sodium deoxycholatedissolved in 10 mM HEPES buffer with 10 mM EDTA (pH 7.8).Decellularization solution was changed after 1 hour throughcentrifugation at 500 rpm for 3 minutes. After fresh solution was added,the tissue material was allowed to incubate for another 4 hours.Decellularization solution was drained after another round ofcentrifugation.

(iii) Enzyme treatment. Decellularized tissue flakes were washed twicefor 30 minutes at a time with 10 mM HEPES buffer containing 5 mM EDTA(pH 7.3). Enzyme treatment was carried out for 4 hours in 150 mL ofHEPES buffer with 2 mM MgCl₂, 2 mM CaCl₂, 1 mg/L dornase alfa, and 1mg/L α-galactosidase. Enzyme solution was drained after centrifugationat 500 rpm for 3 minutes.

(iv) Disinfection. Enzyme-treated tissue flakes were re-suspended andwashed twice with 10 mM HEPES buffer with 5 mM EDTA. The first wash wasfor 60 minutes and the second wash was conducted overnight. Aftercentrifugation, the tissue material was rinsed with 100 mL steriledistilled water for 30 minutes. For some bottles, 100 mL isopropylalcohol (IPA) solution (70% w/v) was added to the tissue suspension.After about 10 minutes, the suspension was centrifuged at 500 rpm for 3minutes. Old IPA solution was drained, and fresh 70% IPA solution wasadded to re-suspend the material. The material was treated with IPA forat least 4 to 6 hours before being packaged in Tyvek bags for freezedrying. For other bottles, tissue flakes were directly packaged in Tyvekbags for freeze-drying without IPA disinfection.

(v) Freeze drying. Processed tissue material was freeze-dried.Freeze-drying consisted of 3 stages: (a) cooling the material from roomtemperature to −35° C. at ˜1° C./minute, then holding at −35° C. for 10minutes; (b) ramping up to −10° C. at ˜1° C./minute under 40 mT, thenholding for 16 hours; and (c) ramping up to 20° C. at ˜1° C./minuteunder 20 mT and then holding for 8 hours.

(vi) Sterilization. Freeze-dried tissue samples that were notdisinfected with IPA were sterilized with ethylene oxide (EO). EOsterilization included (a) conditioning at 52° C. to 63° C. and 55 to75% Relative Humidity for 30 to 45 minutes, (b) EO exposure with a gasconcentration of 600±50 mg/L for 4 hours, and (c) aeration at 38 to 54°C. for at least 12 hours.

(vii) Secondary packaging. IPA-treated samples were packaged immediatelyinto foil-to-foil bags after freeze-drying. EO treated samples werepackaged in foil-to-foil bags following EO treatment.

The processed tissue flakes were acellular. Both IPA-treated andEO-treated materials tested to be sterile.

Example 4 Stability of Processed Tissue Flakes

Thermal stability of processed tissue flakes was tested usingdifferential scanning calorimetry (DSC). Both IPA-treated and EO-treatedmaterials were rehydrated in PBS saline (pH 7.5). Samples were scannedat a heating rate of 3° C./min from 2 to 125° C. (DSC Q200, TAInstruments). Both IPA-treated and EO-treated tissue had a small lowtemperature peak (˜30 to 32° C.). As shown in FIG. 7, the onsettemperature of the major collagen denaturation peaks was 53.3±0.2° C.and 58.0±0.2° C. (N=4) for EO-treated tissue flakes and IPA-treatedtissue flakes, respectively. The onset temperature of IPA-treated tissueflakes was similar to the fresh dermis samples. The data indicated thatEO sterilization destabilizes tissue matrix compared to IPA treatment.

The susceptibility of processed, freeze-dried tissue flakes to enzymedegradation was tested with collagenase and trypsin assays. FIGS. 8A and8B show the resistance of processed tissue material to collagenase andtrypsin digestion, respectively. IPA-disinfected tissue flakes resistedcollagenase and trypsin digestion fairly well. EO-sterilized materialhad increased susceptibility to proteolysis compared to theIPA-disinfected material.

Example 5 Suitability of Tissue Flakes to Permit Fluid Flow and PressureTransduction

Upon rehydration, tissue flakes form a stable suspension without gellingor phase separation, allowing the tissue flakes to be used for fillinglarge voids or defects (tens to hundreds of milliliters). The suspensionof tissue flakes contains large, inter-connected channels that permitfluid to flow freely, and thus aid cell repopulation andrevascularization when the flakes are used as tissue fillers. Theability of the tissue flake suspension to permit the flow of fluids andthe transduction of pressure differentials was investigated. Rehydratedtissue flakes were placed onto Organza mesh and spread out over a 3″×3″pressure distribution pad with 36 sensor ports, as shown in FIGS. 9A and9B. A piece of GranuFoam™ (3″×3″, and 1.5″ thick) was placed on the topof the tissue flake material and a 6″×6″ V.A.C.® was attached to thedressing assembly along with a T.R.A.C.® pad. The assembly was connectedto the V.A.C.® ATS therapy unit to produce a continuous pressure at 125mm Hg. After 5 minutes of equilibrium, the negative pressure detected atthe 36 ports was noted. Thereafter, 4 of the ports were disconnectedfrom the pressure sensors and connected to a reservoir of dyed 0.9%saline solution for infusion at a rate of 500 mL per day via aperistaltic pump. The pressure at the 32 remaining ports was thenmonitored over time. The average pressure detected was approximately110-112 mm Hg. The negative pressure was 111.4±0.9 (mean±SD), 110.1±1.4,and 110.6±1.3 mm Hg after 1, 2, and 3 hours respectively. Theconsistency indicated that pressure was distributed evenly across theentire dressing assembly, and the inter-connected channels were able totransduce pressure differentials well.

Example 6 Reconstructive Tissue Foam Made From Flake-Like TissueMaterial

an acellular tissue matrix derived from porcine dermis was frozen at−80° c. and used to make tissue flakes aseptically with a medium sizeMICROPLANE® cheese grater. Approximately 50 g of tissue sample wassuspended in 200 mL sterile water and then mixed using a RETSCH® blenderat 4000 rpm in one minute intervals for a total of 5 cycles. As shown inFIGS. 10A and 10B, blending reduced the size of the tissue flakematerial. Blending the tissue flakes also resulted in a stable andconsistent tissue suspension. The tissue suspension was distributed in80 cm² plastic petri dishes at 25 mL suspension per dish andfreeze-dried aseptically. The freeze-drying process included thecontrolled cooling of the tissue suspension from room temperature to−30° C. within 60 minutes and drying at a chamber pressure of 100 mT anda shelf temperature of 20° C. for 24 hours.

The process of blending and freeze-drying resulted in small tissuepieces of various dimensions intertwined and interlocked with oneanother, forming a soft tissue foam with interconnected macropores andpreserved extracellular tissue matrix structures, as shown in FIGS. 11Athrough 11D. FIG. 11A presents a gross appearance of the tissue foam.FIG. 11B shows the interconnected macropores of the soft tissue foamunder SEM microscopy. FIG. 11C is a histogram of the extracellularmatrix structure of the foam using a hematoxylin and eosin stain. FIG.11D is an enlarged view of a section found within FIG. 11C. As shown inFIGS. 11C and 11D, the tissue matrix of the foam has a fibrous,filamentous nature after blending and freeze-drying. The asepticallyfreeze-dried foam had a dry tissue mass of 9.9±0.3% (w/v, N=5).

Some of the freeze-dried tissue material was further treated at ˜100° C.under vacuum for 24 hours to increase the strength of the tissue foam.calorimetric measurement detected an onset denaturation temperature of62.2±0.1° C. and a denaturation enthalpy of 60.5 J/g tissue mass,indicating no tissue collagen denaturation.

In a separate experiment using the freeze-dried material, the in vivoresponse of reconstructive tissue foam made from flake-like tissuematerial was investigated using an athymic rat model (Rattus norvegicus,nude rat). Tissue specimens (10 mm×10 mm, and ˜3 mm thick) were preparedfrom the tissue foam and rehydrated in 0.9% saline solution. Afterrehydration, the tissue specimens were then implanted subcutaneously inathymic rats. For each rat, four separate incisions were made on theright and left side of the back through the skin and parallel to thelumbar region of the vertebral column. Pockets were formed by bluntdissection in the subcutaneous tissue in which the tissue material wasintroduced. Two specimens were implanted on the right side of thevertebral column and two specimens were implanted on the right, with theskin closed thereafter. Animals were euthanized either four or eightweeks after implantation. Implanted specimens with attached adjacentsoft tissue were excised and the excised tissue samples were fixated in10% formalin and processed for histological evaluation using hematoxylinand eosin stains. Histological slides were assessed microscopically forevidence of host cell repopulation and re-vascularization. As shown inFIG. 12, significant cell repopulation and revascularization wereobserved in 4-week implants (FIGS. 12A and 12B), while implanted tissuefoams were fully repopulated in 8-weeks (FIGS. 12C and D). Inflammationwas observed to be mild in 4-week explants, and subdued in 8-weekexplants.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1. A method for preparing a tissue matrix composition comprising:selecting a collagen-based tissue matrix; contacting the matrix with acryoprotectant solution; freezing the matrix; and cutting the matrix,wherein the temperature of the tissue matrix ranges from −10° C. to −40°C. for the cutting step.
 2. The method of claim 1, wherein the tissuematrix comprises an acellular tissue matrix.
 3. The method of claim 2,wherein the acellular tissue matrix comprises an acellular dermalmatrix.
 4. The method of claim 2, wherein the acellular tissue matrixcomprises an acellular porcine dermal matrix.
 5. The method of claim 1,wherein the cryoprotectant solution comprises at least one of amaltodextrin, sucrose, polyethylene glycol (PEG), orpolyvinylpyrrolidone (PVP) solution, or a combination thereof.
 6. Themethod of claim 1, wherein the cryoprotectant solution comprises amaltodextrin solution.
 7. The method of claim 6, wherein themaltodextrin solution comprises 5-50% (w/v) maltodextrin.
 8. The methodof claim 6, wherein the maltodextrin solution comprises 15-25% (w/v)maltodextrin.
 9. The method of claim 1, wherein cutting comprisesgrating.
 10. The method of claim 1, wherein the tissue matrix is cutwith a grater.
 11. The method of claim 1, wherein the tissue matrix iscut with a grating wheel.
 12. The method of claim 1, wherein the cuttissue has a size distribution ranging from 0.2-5 mm in length, 0.2-3 mmin width, and 0.02-0.3 mm in thickness.
 13. The method of claim 1,further comprising exposing the tissue matrix to a disinfectant.
 14. Themethod of claim 13, wherein the disinfectant is isopropyl alcohol. 15.The method of claim 1, further comprising sterilizing the tissue matrix.16. The method of claim 15, wherein sterilizing the tissue matrixcomprises application of ethylene oxide, propylene oxide, gammairradiation or e-beam irradiation.
 17. The method of claim 1, furthercomprising lyophilizing the tissue matrix.
 18. The method of claim 1,wherein the cut tissue comprises pieces of cut tissue having irregularsize and shape.
 19. The method of claim 1, further comprising adding abioactive substance to the cut matrix.
 20. The method of claim 19,wherein the bioactive substance comprises an antimicrobial agent. 21.The method of claim 19, wherein the bioactive substance comprises acytokine.
 22. The method of claim 19, wherein the bioactive substancecomprises a growth factor.
 23. The method of claim 19, wherein thebioactive substance comprises non-collagenous tissue.
 24. The method ofclaim 23, wherein the non-collagenous tissue comprises adipose tissue.25. The method of claim 19, wherein the bioactive substance comprisescells.
 26. The method of claim 25, wherein the cells comprise stemcells. 27.-105. (canceled)